A total absence of enteric ganglia in the distal portions of the intestine is known as Hirschsprung disease (HSCR). Children with HSCR present with a constellation of symptoms (e.g., severe constipation, diarrhea, intestinal inflammation) that predispose to sepsis, and can be life-threatening. Surgical correction eradicates these symptoms in most cases. But in up to 30% of cases, surgery fails to cure the disorder, and thereafter, children suffer from chronic enterocolitis that can lead to bacteremia, outright sepsis, and even death. Almost nothing is known about the underlying mechanisms of this enterocolitis, known as Hirschsprung-associated enterocolitis (HAEC). In the course of our detailed studies of children with HSCR who developed HAEC, we discovered a striking shift in fungal microflora diversity with increased Candida albicans in the stool of children with a history of HAEC (but not active HAEC), compared with those who did not develop HAEC (Frykman et al. PloS One 10:e0124172, 2015). The dramatic shifts in fungal diversity in HAEC patients could potentially be persistent fungal blooms as a consequence of multiple courses of antibiotics aimed at treating the enterocolitis. Alternatively, there may be early changes in the bacterial and fungal microbiome that occur as a result of colonic aganglionosis that predispose children to HAEC or potentially altered innate immunity such that C albicans may be a pathobioant. Regardless of which possibility may be correct, the individual contributions made by colonic bacteria and fungi to the development of enterocolitis in children with Hirschsprung disease remains unclear. Endothelin Receptor B gene (Ednrb) mutations cause colonic aganglionosis in both mice and humans (Hirschsprung disease) that develop severe enterocolitis. The mechanisms that contribute to HAEC are poorly understood, and the current treatment approaches are directed towards control of symptoms, with very limited success. Hence, treatment with antifungal agents could be novel and targeted therapy to treat children with HAEC, who currently have few alternatives. Our overall hypothesis is that fungi contribute to the dysbiosis of HSCR children who develop HAEC, and the Ednrb-null mice that develops enterocolitis serves as an appropriate and clinically relevant model to study microbiomal changes. Preliminary data indicate that Ednrb-null mice with enterocolitis have increased gut C. albicans. Our plan is to first comprehensively characterize the bacterial and fungal microbiome, especially the development of enterocolitis, in Ednrb-/- mice with colonic aganglionosis; then to identify the functional role of colonic bacteria, fungi and innate immunity in enterocolitis. Our ultimate goal is to understand the role of fungi in tissue inflammation of HAEC, which can best be studied in a mouse model. This R03 application proposes a detailed strategy to pursue these mechanistic studies related to HAEC, while still under the guidance of his outstanding K08 mentoring team. The ultimate goal is for the applicant to leverage the data obtained during this proposed investigation along with human data, into a successful R01 application.